Project Summary DNA mismatch repair (MMR) plays critical roles in eukaryotic cells including: 1) suppressing mutations that result from misincorportation errors during DNA replication that escape proofreading; 2) suppressing mutations due to mispairs that result from misincorporation events that occur in response to chemical modification of DNA or DNA precursors; 3) preventing genome rearrangements due to recombination between divergent DNA sequences; 4) correcting mispaired bases in heteroduplex recombination intermediates; and 5) detecting DNA damage and activating signaling pathways linked to cellular responses, including cell cycle control and cell death. Consequently, MMR defects cause increased rates of accumulating mutations and genome rearrangements resulting in a characteristic genome instability signature and resistance to killing by some DNA damaging agents. In humans, MMR defects underlie both inherited and sporadic cancers and cause tumors to become resistant to some chemotherapy agents but appear to cause sensitivity of cancers to immunotherapy. Thus, a better understanding of MMR pathways and the consequences of MMR defects will impact human health by: 1) informing the development and improvement of clinical tests for MMR status; and 2) guiding improvements in the development and use of therapies for MMR-deficient cancers. The proposed studies use Saccharomyces cerevisiae as a model system to study the mechanisms of the conserved eukaryotic MMR pathways. The following lines of investigation will be carried out: 1) genetic approaches will be used to study the Exo1-dependent and -independent MMR sub-pathways focusing on Msh2- and Mlh1-interacting proteins and identifying mutations in the MSH2 and MSH6 genes that specifically inactivate Exo1-dependent or Exo1-independent MMR for use in biochemical studies of MMR; 2) the biochemical properties of individual MMR proteins will be characterized to understand the roles that each protein plays in MMR focusing on Exo1, the Mlh1-Mlh3 complex and MMR sub-pathway-specific roles of the Msh2-Msh6 complex; 3) reconstitution approaches will be used to study Exo1-independent MMR, the role of Mlh1-Pms1 in Exo1-mediated mispair excision, and replication-coupled MMR; and, 4) individual steps in MMR reactions will be studied primarily by investigating the protein-protein interactions that drive MMR and studying how the Mlh1-Pms1 endonuclease is correctly targeted to the DNA strand to be repaired. The long-term goal of these studies is to develop a detailed understanding of the biochemical and molecular mechanisms of MMR and how cells utilize MMR to prevent mutations and genome rearrangements. Because MMR is highly conserved among eukaryotes, the results from studies of S. cerevisiae MMR will provide insights into the mechanisms of MMR in human cells. Consequently, this project will provide insights that can be applied to understanding the genetics of inherited and sporadic human cancers and the biology of MMR defects in human cancers in addition to providing a basic understanding of MMR mechanisms.